The identification of apoptotic events has important applications for detecting cell death induced by disease, injury, therapeutics, or normal tissue development and differentiation. Activation of the intracellular caspase cascade is a well understood mechanism that results in apoptosis. Further advances in medical research increasingly call for in vivo methods and probes for detection and imaging of intracellular signaling events such as the induction of caspases. Immunochemistry Technologies, LLC (ICT) has made fluorochromeconjugated caspase inhibitors for detecting caspase activity in vivo (FLIVOTM) that are modified with a fluoromethyl ketone (FMK). The FMK modification creates an irreversible binding inhibitor. However, the perceived potential for generating toxic fluoroacetates makes these FMK probes undesirable for some investigators: Therefore, Phase I of this SBIR proposes to replace the FMK moiety with a 2,6-difluorophenoxy (OPH) group to eliminate this concern. The newly synthesized OPH-modified fluorescent probes will be compared with the existent FMK-modified probes for their ability to specifically detect caspase activity in vitro and for their suitability as potential in vivo apoptosis detection reagents. Accordingly, the Specific Aims of the Phase I portion of this proposal are: Specific Aim I. Conjugation of FAM-VAD and SR-VAD to OPH and optimization of the conjugation protocol. Specific Aim II. Compare the in vitro detection capability of the OPH-modified probes to the standard FMK-modified probes, and Specific Aim III. Evaluate the potential toxicity of the FMK- and OPH- modified probes. In Phase II of this proposal, the fluorochrome-conjugated FMK- and OPH-modified probes will be evaluated for their comparable ability to detect activated caspases in vivo using five different tumor model systems. The success of ICT in generating these caspase detection probes results in a novel method for in vivo detection of the efficacy of cancer-based therapeutics. The Specific Aims of the Phase II portion of this proposal are: Specific Aim I. Detection of apoptotic tumor cells in mice bearing GL261 gliomas treated with tumor cell lysate and CpG Oligodeoxynucleotide (ODN) vaccine. Specific Aim II. Detection of apoptotic tumor cells in mice bearing EMT6 mammary carcinoma treated with tumor cell lysate and CpG ODN vaccine. Specific Aim III. Detection of apoptotic tumor cells in mice bearing LS 174T colon carcinoma and treated with gamma interferon. Specific Aim IV. Detection of apoptotic tumor cells in mice bearing the B16-F10 melanoma and treated with B16a cell vaccine, and Specific Aim V. Detection of apoptotic tumor cells in mice bearing RM-1 prostate carcinoma and treated with RM-1a cell vaccine. Completion of these studies will add two new caspase detection probes to our line of FLIVOTM products. These studies will provide the necessary data for optimizing the dosing and bottling concentration of all of the product probes, as well as a generalized protocol for their use in vivo. Finally, it is hypothesized that these animal studies will provide a foundation for eventual use in human patients.